Intracellular Ca2+ and pH are two key regulators of the ability of sperm to fertilize an egg. Intracellular Ca2+ and pH are controlled in turn by sperm ion channels. Therefore, to understand the molecular mechanisms that control sperm function and male fertility, we need a more thorough understanding of sperm ion channels. Unfortunately, extreme difficulty in applying the patch-clamp technique to sperm cells has hampered our understanding of sperm ion channels and the molecular mechanisms controlling male fertility. We have overcome this barrier and developed a method to apply the whole-cell patch clamp technique to mouse and human spermatozoa. Surprisingly, our patch-clamp experiments revealed significant differences between ion channels in mouse and human spermatozoa. These differences indicate the potential pitfalls of relying on animal models for studying human male fertility and support the need to study these ion channels specifically in human sperm cells. Our long-term objective is to elucidate the mechanisms of ion channel-based signaling that control fertility in human spermatozoa. Here we propose three specific aims to expand our knowledge of sperm ion channels. In Specific Aim 1, we will identify the physiological regulators of human CatSper and Hv1 channels. We hypothesize that key regulators of sperm activity, such as progesterone, prostaglandins, cholesterol, and cAMP, are likely to mediate their actions on human spermatozoa by regulating CatSper or Hv1 channels. We will use the patch-clamp technique to test the effects of the above mentioned compounds on currents mediated by CatSper and Hv1 channels. In Specific Aim 2, we will identify the membrane (non- genomic) progesterone receptor of human spermatozoa. Our preliminary results have identified a narrow group of specific proteins as candidates for the sperm progesterone receptor. We will determine which of the candidate proteins serves as a progesterone receptor and will identify its ligand-binding domain for progesterone. In Specific Aim 3, we will characterize the acrosomal ion channels of human spermatozoa. We will develop a method for applying the patch-clamp technique to the acrosome of human spermatozoa and then use this method to characterize acrosomal Ca2+ channels that are likely to release Ca2+ from the acrosome and to identify the mechanisms that regulate their activity. The knowledge gained from this research will help us to understand the causes of male infertility and to develop new approaches for infertility treatment as well as contraception.